CN103433484A - Bovine serum albumin-platinum composite nanomaterial mimetic peroxidase - Google Patents
Bovine serum albumin-platinum composite nanomaterial mimetic peroxidase Download PDFInfo
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Abstract
The invention discloses bovine serum albumin-platinum composite nanomaterial mimetic peroxidase. Bovine serum albumin is used as a template, and the bovine serum albumin-platinum composite nanomaterial mimetic peroxidase is prepared through biomineralization. Bovine serum albumin-platinum composite nanomaterials are prepared through the following method that chloroplatinic acid aqueous solutions are added to bovine serum albumin aqueous solutions and are mixed, sodium hydroxide aqueous solutions are added to obtain mixed solutions, and water bath heating is carried out; ultrafiltration is carried out on the solutions, then the solutions are washed, and bovine serum albumin-platinum composite nanomaterial aqueous solutions are obtained. The bovine serum albumin-platinum composite nanomaterials have excellent peroxidase activity, and can catalyze hydrogen peroxide oxidation 3, 3', 5, 5'-tetramethyl benzidine hydrochloride to be in color development. Meanwhile, the mimetic peroxidase resists acid and base, high temperature and high salinity, and has excellent short-term indoor temperature stability and long-term indoor temperature stability.
Description
Technical field
The present invention relates to have the bovine serum albumin(BSA) of simulation peroxidase activity-platinum composite nano materials, belong to nanometer technology and bionics techniques field.
Background technology
Enzyme is extremely important in organism, the protein with catalytic activity, and its catalytic efficiency is high, selectivity is strong, reaction condition is gentle, and the vital movements such as the growth of organism, growth, breeding all be unable to do without the catalytic action of enzyme.Yet the native enzyme source is limited, purification difficult is expensive.Simultaneously, native enzyme easily is subject to the impact of multiple physics, chemical factor and loses activity, thus comparatively harsh to the operating condition of experiment in actual applications, its application is greatly limited.In recent years, the development and application of manual simulation's enzyme has been subject to people's extensive concern.
Peroxidase is the catalytic oxidation hydrogen peroxide efficiently, and hydrogen peroxide is a kind of important intermediate material in biological respinse, so the Accurate Measurement of hydrogen peroxide and relevant biochemical substances is had great importance.The related material of Mimetic Peroxidase comprises ferriferrous oxide nano-particle, hemoglobin, hemin, metalloporphyrin, metal phthalocyanine etc.Wherein, the nano artificial analogue enztme has simple, economic, quick, the high temperature resistant and many advantages such as acid and alkali-resistance, stable in properties of preparation, aspect the simulation biology enzyme, is demonstrating extremely tempting application prospect.
The invention provides a kind of high activity, high stability simulation peroxidase based on bovine serum albumin(BSA)-platinum composite nano materials.
Summary of the invention
The objective of the invention is take bovine serum albumin(BSA) as template, by biomineralization, prepare bovine serum albumin(BSA)-platinum composite nano materials, utilize good catalysis characteristics simulation peroxidase of nanoscale metal platinum kernel and its preparation method and application, utilize the bovine serum albumin(BSA) shell to play stabilization to the analogue enztme activated centre.
To achieve these goals, the present invention is by the following technical solutions:
(1) of the present invention a kind of
bovine serum albumin(BSA)-platinum composite nano materials simulation peroxidase is prepared from by following method:add chloroplatinic acid aqueous solution in Bovine Serum Albumin in Aqueous Solution, after mixing, add sodium hydrate aqueous solution to obtain mixed solution, heating water bath, three absworption peaks disappearances (the chloroplatinic acid total overall reaction now is described) of mixed solution its chloroplatinic acid after adding thermal response; This solution, through ultrafiltration washing, is obtained to nano platinum particle-bovine serum albumin(BSA) nucleocapsid structure aqueous solution; The simulation peroxidase activity is characterized as
:add successively hydrogen peroxide, TMB hydrochloride and bovine serum albumin(BSA)-platinum composite nano materials aqueous solution in phosphate buffer, after mixing, temperature is bathed, and solution, by the colourless blueness that becomes, has an absworption peak at 652 nm places.
The proportioning of each component in the preparation of the above-mentioned bovine serum albumin(BSA) of the present invention-platinum composite nano materials can complete by this area conventional method.But prepared by following methods by the preparation of preferred bovine serum albumin(BSA) of the present invention-platinum composite nano materials: add 16 mmol/L platinum acid chloride solution 5 ml and 1.5 mol/L sodium hydroxide solution 0.5 ml in the Bovine Serum Albumin in Aqueous Solution of 5 ml 50 mg/ml, mix reaction 2 h under 80 ℃ of rear water-baths.After reaction, solution is packed into and is ended the super filter tube that molecular weight is 3k, 6000 r/min centrifugal ultrafiltrations, and wash 3 times.
The preferred testing procedure of simulation peroxidase activity feature is
:at 2780 μ L pH=4.5, adding successively hydrogen peroxide, the 0.2 ml concentration that 1 ml concentration is 2 mol/L in the phosphate buffer of 20 mmol/L is 3 of 16 mmol/L, 3 ', 5, the bovine serum albumin(BSA) that 5 '-tetramethyl biphenyl amine hydrochlorate and 20 μ L concentration are 0.36 mmol/L-platinum composite nano materials aqueous solution, mix rear 45 ℃ of temperature and bathe 10 minutes, solution is by the colourless blueness that becomes.
Described bovine serum albumin(BSA)-platinum composite nano materials aqueous solution can obtain bovine serum albumin(BSA)-platinum composite nano materials powder by freeze drying.The all glasswares that use in above process all soak through chloroazotic acid, and thoroughly clean with distilled water, dry.
the preparation method of bovine serum albumin(BSA) of the present invention-platinum composite nano materials simulation peroxidase, comprise the steps:add chloroplatinic acid aqueous solution in Bovine Serum Albumin in Aqueous Solution, after mixing, add sodium hydrate aqueous solution to obtain mixed solution, heating water bath, mixed solution three absworption peaks of its chloroplatinic acid after adding thermal response disappear; This solution, through ultrafiltration washing, is obtained to nano platinum particle-bovine serum albumin(BSA) nucleocapsid structure aqueous solution.
(2) peroxidase activity of bovine serum albumin(BSA) of the present invention-platinum composite nano materials
Of the present invention
a kind of bovine serum albumin(BSA)-platinum composite nano materials simulation peroxidase, can the colour developing of catalyzing hydrogen peroxide oxidation TMB hydrochloride.Specifically, by bovine serum albumin(BSA)-platinum composite nano materials catalysis peroxidase substrate TMB hydrochloride, produce blue substrate, checking and its peroxidase activity of comparison.Add successively hydrogen peroxide, 3 in phosphate buffer, 3 ', 5,5 '-tetramethyl biphenyl amine hydrochlorate and bovine serum albumin(BSA)-platinum composite nano materials aqueous solution, after mixing, temperature is bathed 10 minutes, the variation of visualization color or measure the absorbance (A at 652 nm wavelength places
652).Compare peroxidase activity according to solution colour or by the absorbance calibration curve.
Bovine serum albumin(BSA) of the present invention-platinum composite nano materials
the simulation peroxidasecatalyzing hydrogen peroxide oxidation 3,3 ', 5,5 '-tetramethyl biphenyl amine hydrochlorate generates blue product, this product has maximum absorption band or bovine serum albumin(BSA)-platinum composite nano materials catalyzing hydrogen peroxide oxidation 3 at 652 nm places, 3 ', 5, it is maximum that the activity of 5 '-tetramethyl biphenyl amine hydrochlorate reaches at pH=4.5,45 ℃ the time.
Bovine serum albumin(BSA) of the present invention-platinum composite nano materials
the simulation peroxidasemichaelis constant to the TMB hydrochloride is 0.054 mmol/L, to the Michaelis constant of hydrogen peroxide, is 14.18 mmol/L.
Bovine serum albumin(BSA) of the present invention-platinum composite nano materials
the simulation peroxidasepreserve catalytic activity after 2 hours and have no significant change at 4 ~ 95 ℃ of temperature, there is good stability or after pH 2 ~ 12 preserves 2 hours catalytic activity have no significant change, there is good stability.
Bovine serum albumin(BSA) of the present invention-platinum composite nano materials
the simulation peroxidasepreserve catalytic activity after 2 hours and, still without significant change, have good stability or have good short-term room temperature stability in 2 mol/L sodium chloride solutions, in 24 hours, catalytic activity is without significant change.
Bovine serum albumin(BSA) of the present invention-platinum composite nano materials
the simulation peroxidasehave good short-term room temperature stability, in 24 hours, catalytic activity is without significant change.Have good long-time stability, after at room temperature preserving 30 days, relative catalytic activity can reach 98.9%.
advantage of the present invention:
(1) preparation method used in the present invention is easy fast, without reducing agent.
(2) bovine serum albumin(BSA) in the present invention-platinum composite nano materials has good peroxidase activity.
(3) bovine serum albumin(BSA) in the present invention-platinum composite nano materials good stability, peroxidase activity is subject to the impact of storage temperature, pH value and ionic strength little.
(4) the present invention has good long-time stability, and after at room temperature preserving 30 days, relative catalytic activity can reach 98.9%.
The accompanying drawing explanation
The uv absorption spectra that Fig. 1 is bovine serum albumin(BSA)-platinum composite nano materials catalyzing hydrogen peroxide oxidation TMB hydrochloride color development system.
Fig. 2 is the affect figure of bovine serum albumin(BSA)-platinum composite nano materials final concentration on catalystic converter system.
Fig. 3 is the affect figure of pH value on bovine serum albumin(BSA)-platinum composite nano materials catalyzing hydrogen peroxide oxidation TMB hydrochloride color development system.
Fig. 4 is the affect figure of warm bath temperature on bovine serum albumin(BSA)-platinum composite nano materials catalyzing hydrogen peroxide oxidation TMB hydrochloride color development system.
Fig. 5 is the stability kinetics curve map of bovine serum albumin(BSA)-platinum composite nano materials for the TMB hydrochloride.
Fig. 6 is the stability kinetics curve map of bovine serum albumin(BSA)-platinum composite nano materials for hydrogen peroxide.
Fig. 7 is the affect figure of storage temperature on bovine serum albumin(BSA)-platinum composite nano materials catalytic activity.
Fig. 8 is for preserving the affect figure of pH value on bovine serum albumin(BSA)-platinum composite nano materials catalytic activity.
Fig. 9 is for preserving the affect figure of salinity on bovine serum albumin(BSA)-platinum composite nano materials catalytic activity.
The short-term room temperature stability curve map that Figure 10 is bovine serum albumin(BSA)-platinum composite nano materials.
The specific embodiment
example 1:
Add 16 mmol/L platinum acid chloride solution 5 ml and 1.5 mol/L sodium hydroxide solution 0.5 ml in the Bovine Serum Albumin in Aqueous Solution of 5 ml 50 mg/ml, mix reaction 2 h under 80 ℃ of rear water-baths.After reaction, solution is packed into and is ended the super filter tube that molecular weight is 3k, 6000 r/min centrifugal ultrafiltrations, and wash 3 times.After ultrafiltration, the freeze drying of gained solution obtains bovine serum albumin(BSA)-platinum composite nano materials powder.
example 2:
At 2780 μ L phosphate buffer (pH=4.5,20 mmol/L) adding successively hydrogen peroxide, the 0.2 ml concentration that 1 ml concentration is 2 mol/L in is 3 of 16 mmol/L, 3 ', 5, the bovine serum albumin(BSA) that 5 '-tetramethyl biphenyl amine hydrochlorate and 20 μ L concentration are 0.36 mmol/L-platinum composite nano materials aqueous solution, mix rear 45 ℃ of temperature and bathe 10 minutes, solution, by the colourless blueness that becomes, has an absworption peak (Fig. 1) at 652 nm places.
example 3:
At 2780 μ L phosphate buffer (pH=4.5,20 mmol/L) adding successively hydrogen peroxide, the 0.2 ml concentration that 1 ml concentration is 2 mol/L in is 3 of 16 mmol/L, 3 ', 5, the bovine serum albumin(BSA) of 5 '-tetramethyl biphenyl amine hydrochlorate and 20 μ L variable concentrations-platinum composite nano materials aqueous solution, mix rear 45 ℃ of temperature and bathe 10 minutes, measure 652 nm wavelength place absorbances.As seen from Figure 2, the absorbance of colour developing product increases and increases along with bovine serum albumin(BSA)-platinum composite nano materials final concentration.
example 4:
Adding successively hydrogen peroxide, the 0.2 ml concentration that 1 ml concentration is 2 mol/L in the phosphate buffer (20 mmol/L) of the different pH values of 2780 μ L is 3 of 16 mmol/L, 3 ', 5, the bovine serum albumin(BSA) that 5 '-tetramethyl biphenyl amine hydrochlorate and 20 μ L concentration are 0.36 mmol/L-platinum composite nano materials aqueous solution, mix rear 45 ℃ of temperature and bathe 10 minutes, measure 652 nm wavelength place absorbances.As seen from Figure 3, the relative catalytic activity of bovine serum albumin(BSA)-platinum composite nano materials reaches maximum when pH=4.5.
example 5:
At 2780 μ L phosphate buffer (pH=4.5,20 mmol/L) adding successively hydrogen peroxide, the 0.2 ml concentration that 1 ml concentration is 2 mol/L in is 3 of 16 mmol/L, 3 ', 5, the bovine serum albumin(BSA) that 5 '-tetramethyl biphenyl amine hydrochlorate and 20 μ L concentration are 0.36 mmol/L-platinum composite nano materials aqueous solution, after mixing under different temperatures temperature bathe 10 minutes, measure 652 nm wavelength place absorbances.As seen from Figure 4, the relative catalytic activity of bovine serum albumin(BSA)-platinum composite nano materials reaches maximum when temperature is 45 ℃.
example 6:
At 2780 μ L phosphate buffer (pH=4.5,20 mmol/L) adding successively hydrogen peroxide, the 0.2 ml concentration of 1 ml variable concentrations in is 3 of 16 mmol/L, 3 ', 5, the bovine serum albumin(BSA) that 5 '-tetramethyl biphenyl amine hydrochlorate (TMB) and 20 μ L concentration are 0.36 mmol/L-platinum composite nano materials aqueous solution, mix rear 45 ℃ of temperature and bathe 1 minute, measure the absorbance at 652 nm wavelength places.By the Michaelis-Menten equation matching, can show that bovine serum albumin(BSA)-platinum composite nano materials is 0.054 mmol/L(Fig. 5 to the Michaelis constant of TMB).
example 7:
At 2780 μ L phosphate buffer (pH=4.5,20 mmol/L) add successively 3 of hydrogen peroxide that 1 ml concentration is 2 mol/L, 0.2 ml variable concentrations in, 3 ', 5, the bovine serum albumin(BSA) that 5 '-tetramethyl biphenyl amine hydrochlorate and 20 μ L concentration are 0.36 mmol/L-platinum composite nano materials aqueous solution, mix rear 45 ℃ of temperature and bathe 1 minute, measure the absorbance at 652 nm wavelength places.By the Michaelis-Menten equation matching, can show that bovine serum albumin(BSA)-platinum composite nano materials is 14.18 mmol/L(Fig. 6 to the Michaelis constant of hydrogen peroxide).
example 8:
Bovine serum albumin(BSA)-platinum composite nano materials aqueous solution is placed in to preservation under different temperatures and, after 2 hours, measures the impact of storage temperature on its relative catalytic activity.At 2780 μ L phosphate buffer (pH=4.5,20 mmol/L) adding successively hydrogen peroxide, the 0.2 ml concentration that 1 ml concentration is 2 mol/L in is 3 of 16 mmol/L, 3 ', 5, the bovine serum albumin(BSA) that 5 '-tetramethyl biphenyl amine hydrochlorate and 20 μ L concentration are 0.36 mmol/L-platinum composite nano materials aqueous solution, mix rear 45 ℃ of temperature and bathe 10 minutes, measure the absorbance at 652 nm wavelength places.As seen from Figure 7, bovine serum albumin(BSA)-platinum composite nano materials is preserved catalytic activity after 2 hours and is had no significant change at 4 ~ 95 ℃ of temperature, has good stability.
example 9:
Bovine serum albumin(BSA)-platinum composite nano materials aqueous solution is placed in to preservation under condition of different pH and, after 2 hours, measures the impact of storage temperature on its relative catalytic activity.At 2780 μ L phosphate buffer (pH=4.5,20 mmol/L) adding successively hydrogen peroxide, the 0.2 ml concentration that 1 ml concentration is 2 mol/L in is 3 of 16 mmol/L, 3 ', 5, the bovine serum albumin(BSA) that 5 '-tetramethyl biphenyl amine hydrochlorate and 20 μ L concentration are 0.36 mmol/L-platinum composite nano materials aqueous solution, mix rear 45 ℃ of temperature and bathe 10 minutes, measure the absorbance at 652 nm wavelength places.As seen from Figure 8, bovine serum albumin(BSA)-platinum composite nano materials catalytic activity after pH 2 ~ 12 preserves 2 hours has no significant change, and has good stability.
example 10:
Bovine serum albumin(BSA)-platinum composite nano materials aqueous solution is placed in to preservation under variable concentrations sodium chloride condition and, after 2 hours, measures the impact of storage temperature on its relative catalytic activity.At 2780 μ L phosphate buffer (pH=4.5,20 mmol/L) adding successively hydrogen peroxide, the 0.2 ml concentration that 1 ml concentration is 2 mol/L in is 3 of 16 mmol/L, 3 ', 5, the bovine serum albumin(BSA) that 5 '-tetramethyl biphenyl amine hydrochlorate and 20 μ L concentration are 0.36 mmol/L-platinum composite nano materials aqueous solution, mix rear 45 ℃ of temperature and bathe 10 minutes, measure the absorbance at 652 nm wavelength places.As seen from Figure 9, bovine serum albumin(BSA)-platinum composite nano materials is preserved catalytic activity after 2 hours and, still without significant change, is had good stability in up to 2 mol/L sodium chloride solutions.
example 11:
After bovine serum albumin(BSA)-platinum composite nano materials aqueous solution is placed under room temperature and preserves different time, measure its relative catalytic activity.At 2780 μ L phosphate buffer (pH=4.5,20 mmol/L) adding successively hydrogen peroxide, the 0.2 ml concentration that 1 ml concentration is 2 mol/L in is 3 of 16 mmol/L, 3 ', 5, the bovine serum albumin(BSA) that 5 '-tetramethyl biphenyl amine hydrochlorate and 20 μ L concentration are 0.36 mmol/L-platinum composite nano materials aqueous solution, mix rear 45 ℃ of temperature and bathe 10 minutes, measure the absorbance at 652 nm wavelength places.As seen from Figure 10, bovine serum albumin(BSA)-platinum composite nano materials has good short-term room temperature stability, and in 24 hours, catalytic activity is without significant change.
example 12:
Bovine serum albumin(BSA)-platinum composite nano materials aqueous solution is placed in to preservation under room temperature and, after 30 days, measures its relative catalytic activity.At 2780 μ L phosphate buffer (pH=4.5,20 mmol/L) adding successively hydrogen peroxide, the 0.2 ml concentration that 1 ml concentration is 2 mol/L in is 3 of 16 mmol/L, 3 ', 5, the bovine serum albumin(BSA) that 5 '-tetramethyl biphenyl amine hydrochlorate and 20 μ L concentration are 0.36 mmol/L-platinum composite nano materials aqueous solution, mix rear 45 ℃ of temperature and bathe 10 minutes, measure the absorbance at 652 nm wavelength places.Result shows that bovine serum albumin(BSA)-platinum composite nano materials has good long-time stability, and after at room temperature preserving 30 days, relative catalytic activity can reach 98.9%.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any modifications of doing within the spirit and principles in the present invention, be equal to replacement and improvement etc., within all should being included in protection scope of the present invention.
Claims (10)
1. one kind
bovine serum albumin(BSA)-platinum composite nano materials simulation peroxidase, itsbovine serum albumin(BSA)-platinum composite nano materials
by following method, be prepared from:add chloroplatinic acid aqueous solution in Bovine Serum Albumin in Aqueous Solution, after mixing, add sodium hydrate aqueous solution to obtain mixed solution, heating water bath; This solution, through ultrafiltration washing, is obtained to bovine serum albumin(BSA)-platinum composite nano materials aqueous solution; The simulation peroxidase activity is characterized as
:add successively hydrogen peroxide, TMB hydrochloride and bovine serum albumin(BSA)-platinum composite nano materials aqueous solution in phosphate buffer, after mixing, temperature is bathed, and solution, by the colourless blueness that becomes, has an absworption peak at 652 nm places.
2. according to claim 1
a kind of bovine serum albumin(BSA)-platinum composite nano materials simulation peroxidaseit is characterized in that at 2780 μ L pH=4.5, adding successively hydrogen peroxide, the 0.2 ml concentration that 1 ml concentration is 2 mol/L in the phosphate buffer of 20 mmol/L is 3 of 16 mmol/L, 3 ', 5, the bovine serum albumin(BSA) that 5 '-tetramethyl biphenyl amine hydrochlorate and 20 μ L concentration are 0.36 mmol/L-platinum composite nano materials aqueous solution, mix rear 45 ℃ of temperature and bathe 10 minutes, and solution is by the colourless blueness that becomes.
3. according to claim 1 and 2
a kind of bovine serum albumin(BSA)-platinum composite nano materials simulation peroxidaseit is characterized in that prepared by following methods by described bovine serum albumin(BSA)-platinum composite nano materials: add 16 mmol/L platinum acid chloride solution 5 ml and 1.5 mol/L sodium hydroxide solution 0.5 ml in the Bovine Serum Albumin in Aqueous Solution of 5 ml 50 mg/ml, mix reaction 2 h under 80 ℃ of rear water-baths; After reaction, solution is packed into and is ended the super filter tube that molecular weight is 3k, 6000 r/min centrifugal ultrafiltrations, and wash 3 times.
4. one kind
the preparation method of bovine serum albumin(BSA)-platinum composite nano materials simulation peroxidase, comprise the steps:be to add chloroplatinic acid aqueous solution in Bovine Serum Albumin in Aqueous Solution in concentration, after mixing, add sodium hydrate aqueous solution to obtain mixed solution, heating water bath, mixed solution three absworption peaks of its chloroplatinic acid after adding thermal response disappear; This solution, through ultrafiltration washing, is obtained to nano platinum particle-bovine serum albumin(BSA) nucleocapsid structure aqueous solution.
5. claim 1 or 2 or 3 described
a kind of bovine serum albumin(BSA)-platinum composite nano materials simulation peroxidase, the application in the colour developing of catalyzing hydrogen peroxide oxidation TMB hydrochloride.
6. application according to claim 5, is characterized in that catalyzing hydrogen peroxide oxidation TMB hydrochloride generates blue product, and this product has maximum absorption band at 652 nm places; It is maximum that the activity of bovine serum albumin(BSA)-platinum composite nano materials catalyzing hydrogen peroxide oxidation TMB hydrochloride reaches at pH=4.5,45 ℃ the time.
7. according to the described application of claim 5 or 6, it is characterized in that bovine serum albumin(BSA)-platinum composite nano materials is 0.054 mmol/L to the Michaelis constant of TMB hydrochloride, is 14.18 mmol/L to the Michaelis constant of hydrogen peroxide.
8. according to the described application of claim 5 or 6, it is characterized in that at 4 ~ 95 ℃ of temperature or after pH 2 ~ 12 preserves 2 hours catalytic activity have no significant change, there is good stability.
9. according to the described application of claim 5 or 6, it is characterized in that preserving catalytic activity after 2 hours and, still without significant change, there is good stability in 2 mol/L sodium chloride solutions.
10. according to the described application of claim 5 or 6, it is characterized in that having good short-term room temperature stability, in 24 hours, catalytic activity is without significant change; Have good long-time stability, after at room temperature preserving 30 days, relative catalytic activity can reach 98.9%.
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